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Nat Neurosci. Author manuscript; available in PMC 2017 July 11. Published in final edited form as: Nat Neurosci. 2016 May ; 19(5): 690–696. doi:10.1038/nn.4280.

The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability Katharina E Cosker1,3, Sara J Fenstermacher1,3, Maria F Pazyra-Murphy1, Hunter L Elliott2, and Rosalind A Segal1,# 1Department

of Neurobiology, Harvard Medical School, Department of Cancer Biology, DanaFarber Cancer Institute, Boston MA 02215, USA

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2Image

and Data Analysis Core, Harvard Medical School, Boston, MA 02115, USA

Abstract

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To achieve accurate spatiotemporal patterns of gene expression, RNA-binding proteins (RBPs) guide nuclear processing, intracellular trafficking, and local translation of target mRNAs. In neurons, RBPs direct transport of target mRNAs to sites of translation in remote axons and dendrites. However, it is not known whether an individual RBP coordinately regulates multiple mRNAs within these morphologically complex cells. Here we identify SFPQ (Splicing factor, proline-glutamine rich) as an RBP that binds and regulates multiple mRNAs in dorsal root ganglion (DRG) sensory neurons and thereby promotes neurotrophin-dependent axonal viability. SFPQ acts within nuclei, cytoplasm and axons to regulate multiple functionally related mRNAs essential for axon survival. Notably SFPQ is required for co-assembly of laminb2 and bclw within RNA granules and for axonal trafficking of these mRNAs. Together these data demonstrate that SFPQ orchestrates spatial gene expression of a newly identified RNA regulon essential for axonal viability.

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Regulated intracellular trafficking of mRNAs and of ribosomal machinery allows localized translation, and so provides a mechanism for spatiotemporal control of protein expression. Precise mRNA localization is mediated by an array of RNA-binding proteins (RBPs)1. Individual RBPs interact with newly transcribed mRNAs within the nucleus and enable nuclear export2. Subsequently, mRNAs destined for remote subcellular locations are assembled together with interacting RBPs into RNA granules, which are transported by motor proteins along microtubules to sites of local translation1,2. Intriguingly an individual RBP can bind multiple functionally related mRNAs and also coordinate sequential steps of

Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms # Corresponding author: Rosalind Segal, Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Ave, Boston, MA, 02215, 617-632-4737, [email protected]. 3These authors contributed equally to this work AUTHOR CONTRIBUTIONS K.E.C., S.J.F. and R.A.S. designed experiments and wrote the manuscript. K.E.C., S.J.F. and M.F.P.-M. performed compartmented culture experiments. K.E.C. performed formaldehyde cross-linking experiments. M.F.P.-M. performed axonal degeneration and protein transfection experiments. H.L.E performed analysis of FISH data. K.E.C. and S.J.F. performed all other experiments. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

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mRNA processing3,4. In this way, RBPs can synchronize post-transcriptional expression of a set of related mRNAs as an RNA regulon3,4. RNA regulons may be particularly important in highly polarized neurons, where localized mRNAs are translated in axons and dendrites at great distances from the nucleus5,6.

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The DBHS (Drosophila Behavior Human Splicing) family member SFPQ7 is a multifunctional RBP with roles in RNA transcription, splicing and 3′ end processing8–12. SFPQ is highly expressed in the developing and mature nervous system and is required for neuronal survival and normal brain development13,14. Within neurons, SFPQ is a critical component of cytoplasmic RNA transport granules in dendrites15,16. However, it is not known whether SFPQ is required for post-transcriptional regulation of mRNAs that are localized to axons. Recent studies have characterized preferred SFPQ binding motifs, providing a resource for identifying mRNAs that might interact with SFPQ17. Here we demonstrate that SFPQ binds and regulates multiple mRNAs that are localized to the axons of DRG sensory neurons and encode proteins that promote axon survival. We show that SFPQ is required for colocalization of laminb2 and bclw mRNA within cytoplasmic RNA granules and for coordinated axonal trafficking of these mRNAs. Together these studies identify an SFPQ-dependent RNA regulon that coordinates the localization of mRNAs to promote axon survival.

RESULTS SFPQ binds axonal mRNAs

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To determine whether SFPQ coordinates spatial regulation of mRNAs in neurons, we searched for putative SFPQ binding motifs17 within mRNAs that have been demonstrated to localize to axons18. We find numerous SFPQ binding motifs within axonal mRNAs encoding proteins that promote neurotrophin-dependent neuronal survival, including laminb219, bclw20, impa121, and creb22 (Fig. 1a and Supplementary Fig. 1a). These motifs are enriched within the 3′UTRs, which are often critical for proper localization of mRNA1,2 (Fig. 1a and Supplementary Fig. 1a). We tested whether SFPQ binds these functionally related mRNAs by formaldehyde crosslinking and RNA-immunoprecipitation (Supplementary Fig. 1b). Endogenous SFPQ co-precipitates with laminb2, bclw, impa1 and creb mRNA, but not with other functionally distinct axonal mRNAs (Fig. 1a). Furthermore, binding of SFPQ to laminb2, bclw, impa1 and creb is regulated by stimulation with high levels of NGF (Supplementary Fig. 1c). These data demonstrate that SFPQ binds multiple axonal mRNAs that contain SFPQ motifs and are involved in promoting neurotrophindependent survival.

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SFPQ promotes axonal localization of mRNAs We examined the subcellular localization of SFPQ in DRG sensory neurons, as these cells extend long peripheral axons and depend on target-derived neurotrophins for survival23. We detect SFPQ in the peripheral axons that extend towards neurotrophin-producing target tissues (Fig. 1b, c) and in the nerve endings that innervate the skin (Fig. 1d). As an RBP with roles in nuclear RNA processing, SFPQ is also present in nuclei as well as cytoplasm of sensory neurons (Supplementary Fig. 1d). A similar pattern of expression is observed in

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cultured sensory neurons, where SFPQ localizes to distal axons and growth cones, nuclei and surrounding cytoplasm (Fig. 1e and Supplementary Fig. 1e, f, g). The presence of SFPQ in axons of sensory neurons both in vivo and in vitro suggests a potential role for SFPQ in localization of mRNAs to axons. To test this, we used sensory neurons in compartmented cultures that recapitulate the separation of cell bodies and distal axons observed in vivo. In this system, neurotrophin stimulation of distal axons leads to an increase in laminb2, bclw, impa1, creb, and β-actin mRNA levels in axons (Fig. 2a). We find that shRNA-mediated knockdown of SFPQ (Fig. 2e, Supplementary Fig. 2a) inhibits neurotrophin-dependent increases in multiple axonal mRNAs that are bound by SFPQ (Fig. 2a). Importantly, knockdown of SFPQ does not alter the neurotrophin-dependent increase in axonal β-actin mRNA24, which is not bound by SFPQ (Fig. 2a). Collectively these data demonstrate that SFPQ is required for neurotrophin-dependent axonal localization of multiple functionally related genes.

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Both laminb2 and bclw mRNA are bound by SFPQ and loss of either component leads to a similar phenotype of selective axon degeneration in vivo19,25. Furthermore, while LaminB2 was initially identified as a nuclear intermediate filament, and Bclw was discovered as an anti-apoptotic Bcl2 family member, both laminb2 and bclw are locally translated within axons, where the resultant protein products localize to mitochondria19,20. Therefore, in the following experiments, we focused on these two functionally related axonal mRNAs. To examine the role of SFPQ in regulating neurotrophin-dependent axonal localization of laminb2 and bclw mRNA, we performed single-molecule fluorescent in situ hybridization (FISH) after validating the specificity of laminb2 and bclw probes (Supplementary Fig. 2b, c). Puncta of laminb2 and bclw mRNA are evident in distal axons of neurons grown in microfluidic cultures (Fig. 2b). Consistent with qRT-PCR data, laminb2 and bclw mRNA puncta are less numerous than β-actin puncta, (Fig. 2b and Supplementary Fig. 1b) and the density of laminb2 and bclw mRNA puncta in axons increases with neurotrophin stimulation (Fig. 2c and Supplementary Fig. 2d). Furthermore, axonal laminb2 and bclw mRNA puncta decrease following knockdown of SFPQ, while β-actin mRNA puncta in axons are not affected (Fig. 2d and Supplementary Fig. 2e). Together these results demonstrate a specific role for SFPQ in neurotrophin-dependent axonal localization of laminb2 and bclw mRNA.

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Since SFPQ is a component of RNA transport granules13,14, we asked whether impaired localization of laminb2 and bclw mRNA to axons (Fig. 2a) is associated with corresponding changes of mRNA levels within cell bodies. We carried out subcellular fractionation and isolated mRNAs from nuclei, cytoplasm, and distal axons (Fig. 3a, e). Following knockdown of SFPQ (Supplementary Fig. 2a), laminb2 mRNA accumulates in the cytoplasmic fraction (Fig. 3b), suggesting that SFPQ is required for axonal transport of laminb2 mRNA. In contrast, bclw mRNA accumulates in the nuclear fraction following knockdown of SFPQ (Fig. 3c), suggesting SFPQ may be required for both nuclear export and subsequent axonal transport of bclw mRNA. Importantly, subcellular localization of β-actin mRNA is not altered by SFPQ knockdown (Fig. 3d). Together these results indicate that SFPQ functions throughout the neuron to enable axonal trafficking of interacting mRNAs.

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SFPQ is required for mRNA coassembly in transport granules

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It is not known whether distinct mRNAs that bind the same RBP are co-assembled into an individual RNA transport granule. Electron microscopy studies estimate RNA granules to be 100–250 nm in diameter26. To determine whether laminb2 and bclw mRNA are packaged together, we performed single-molecule FISH and looked for colocalization of laminb2 and bclw mRNA in the cell body, where RNA transport granules are packaged for axonal transport (Fig. 4a). Using super-resolution quantitative colocalization analysis, we observe an enrichment of laminb2 and bclw mRNAs localized within a distance

The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability.

To achieve accurate spatiotemporal patterns of gene expression, RNA-binding proteins (RBPs) guide nuclear processing, intracellular trafficking and lo...
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